The goal of this project is to determine the mechanism of B lymphocyte differentiation leading to a diverse immune repertoire and how this process is regulated. This will be accomplished under defined conditions by the use of in vitro lymphocyte culture systems including a fetal organ culture and long term lymphocyte cultures. B cell development and the associated diversity will be assessed by the following criteria: 1) selection and expression of VH and VL gene families at the single cell level by in situ hybridization with radiolabelled VH and VL gene probes, 2) mitogen responsiveness, 3) frequency of antigen specific B cells and associated idiotypes using the splenic fragment assay, and 4) acquisition of B lineage surface markers. Because it is now possible to assess V region expression in the unstimulated B cell or pre B cell, important background information will be obtained. The available VH and VL gene family repertoire, in the absence of stimulation, will be precisely determined at various ages in different tissues, and in different strains. Evidence is presented for distinct B cell progenitors in the adult vs fetus. Experiments outlined will determine the role distinct progenitors plays in B cell formation and the generation of diversity. In addition, the effect of microenvironment on B cell differentiation and repertoire development will be defined using cloned stromal cell lines producing distinct B lineage growth and differentiation factors. The effect of adding exogenous factors will also be determined. Finally, parameters that affect the selection of the B cell repertoire will be assessed. These experiments should provide new insights into B cell differentiation and the generation of diversity, insights which should contribute to an understanding of both cancer and autoimmunity.